Structural investigations of the Get4/Get5/Sgt2 complex

Abstract

Tail‐anchored (TA) proteins are classified by a single transmembrane helix at the extreme carboxyl terminus. In yeast, the Guided Entry of Tail‐anchored proteins (GET) pathway facilitates the post‐translational targeting of TA proteins to the endoplasmic reticulum (ER). A complex consisting of Get4, Get5, Sgt2 and chaperones delivers ER destined substrates to Get3, which then targets the substrate to the ER membrane. We have structurally characterized the Get4/Get5/Sgt2 complex. Get4 is an alpha‐helical repeat protein that directly binds Get3. Get4 and Get5 form an obligate dimer mediated by the amino terminal domain of Get5, and this heterodimer forms a higher order dimer mediated by the carboxyl terminal domain of Get5. We have determined structures of the Get5 homodimerization domain by crystallography and solution NMR and found it is a novel, high‐affinity oligomerization motif. Sgt2 contains a tetratricopeptide repeat (TPR) domain that binds to a wide variety of heat shock cognate (HSC) protein families. A crystal structure of the TPR domain with an HSC‐analog provides an explanation for this low specificity. Sgt2 also contains an amino terminal homodimerization domain that additionally binds a ubiquitin‐like domain within Get5. We utilize bioSAXS to combine high‐resolution structural data, generating a model of the Get4/Get5/Sgt2 complex in solution.